Allergies are elicited by the allergen-mediated-clustering of the immunoglobulin E (IgE) molecules on the surface of mast cells. Allergic diseases are increasing health concerns in developed nations. Depending on the severity of the allergic reaction, the results can vary from a simple itch to anaphylactic shock, which results in 1,500 deaths each year in the US. There are no cures for allergies, and current therapies focus on treatment of acute symptoms or chronic localized immune suppression. Even with the most restricted diets, accidental exposure is very frequent for food allergies, putting patients at riskfor life threatening anaphylaxis. Combined, there is a need for more effective, alternative treatments for IgE-mediated allergic responses. The objective of this application is to establish a detailed understanding of epitope-IgE interactions on mast cell degranulation, and to design selective inhibitors of IgE-mediated allergic responses. Because naturally occurring allergens are typically complex, structurally heterogeneous proteins with multiple allergy-inducing epitopes, it has been a challenge to develop cellular experimental model systems that mimic natural allergic responses. First, we will address this problem by developing a multicomponent experimental model system, which will enable an integrative approach by incorporating epitope heterogeneity and IgE variability to better reflect the complexity of natural allergens. We will us this physiologically more relevant model to establish a detailed understanding of epitope-IgE interactions in mast cell stimulation and its inhibition. Next, we will engineer high avidity heterobivalent inhibitors (HBI) of epitope-IgE interactions for selective inhibition of IgE-mediate allergic reactions. The HBI will be designed to simultaneously target two nearby binding sites located on the Fab domain of an IgE: the antigen binding site; and the not-so-well-known nucleotide binding site (NBS). The simultaneous bivalent binding of the HBI to both sites will provide enhanced avidity over that of the allergen epitope, thereby competitively inhibiting allergen-IgE interaction and mast cell degranulation. The specific aims of this proposal are to: i) develop a multicomponent experimental model system that reflects epitope heterogeneity and IgE antibody variability to establish how epitope-IgE interactions affect mast cell degranulation under physiologically relevant conditions; ii) to engineer heterobivalent inhibitors (HBI) that utilize the not-so-well-known NBS for selective inhibition of IgE clustering on mast cells via partial epitope inhibition; iii) to validate of the multicomponent experimental model system and HBI design in mouse allergy models. The proposed work is innovative because it (i) develops a physiologically relevant experimental model system to study mast cell stimulation and its inhibition, (ii) characterizes the not-so-well-known NBS found on all antibodies, and thereby reveals a unique and underutilized feature present in the antibody structure; (iii) investigates a utility for the conserved NBS, and (iv) brings a novel molecular design approach for selective inhibition of IgE-mediated allergic responses.